Not so long ago now, a certain Doctor Carol
Johnston of Arizona
State University Johnston ’s group.
Well, no. Starch is an esoteric enough field that few people
think of that. And so, I volunteered for the task and designed an experiment to
test the proposition in vitro.
The long and the short of it is this: no, Virginia . No, it does not account for the
mechanism. However, the negative result was never published at my advisor’s
will and the matter was dropped. Because these data would otherwise die in a
notebook never to be opened again and I believe would be of benefit to this
field of investigation, I am putting up the original paper that I wrote at the
time for public viewing here, unaltered from its original state.
The result that we obtained at the time might have been a
definitely negative one, but I am not without an hypothesis as to how the
mechanism occurs. The paper is to be found below the break, and I shall address
what I believe to be a quite probable cause as to the lowering of postprandial
blood sugars by the mere imbibing of vinegar in the next post, for to the best
of my knowledge, the exact nature of the biomolecular interplay has never been
elucidated.
Starch-Acetic Acid Complex Does Not
Significantly Contribute to the Postprandial Glycaemic Lowering Effect of
Vinegar
Introduction
Starch is a primary staple in human diets worldwide. Rapid
digestion of starch results in a steep rise in postprandial blood sugar and
insulin concentrations that may have adverse health consequences if such
elevated levels are chronically sustained, as in diabetes mellitus.
Administration of ingested vinegar prior to meals has been investigated as a
component of treatment for diabetic patients comparable to some drug therapies
(Johnston and others 2004, Liatis and others 2010). The postprandial glycaemic
lowering effect of vinegar was first documented in rats administered a cooked
cornstarch paste containing 0 or 2% acetic acid through intestinal intubation
by Ebihara and Nakajima (1988). Ingestion of vinegar has been shown to
significantly decrease postprandial glycaemia compared with placebo controls in
subjects fed carbohydrate-rich meals incorporating both starch and simple
sugars, including bread (Ostman and others 2005), potatoes with skimmed milk
(Liatis and others 2010), mixed meal with bagel, butter, and orange juice
(Johnston and others 2004), and strawberry vinegar mixed with sucrose (Ebihara
and Nakajima 1988).
Starch is chiefly comprised of two glucose
homopolysaccharides, amylose and amylopectin. Amylose is a primarily linear
molecule made up of α-1,4 lined anhydroglucose units, whereas amylopectin is a
large, highly branched glucose polymer with abundant α-1,6 branch linkages.
Starches from normal botanical sources contain between 20 – 30% amylose, while
waxy starches contain no or very little amylose. Amphiphilic molecules, such as
fatty acids, are known to form helical complexes with starch chains (Ai and
others 2013). Complex formation is governed by hydrophobic interactions of the
hydrophobic moiety of these molecules with the interior hydrophobic cavity of
linear starch chains, namely amylose and long amylopectin branch-chains. Short
amylopectin branch chains, such as those found in cereal starches, exhibit
minimal helical complex formation. Small molecules known to form helical
complexes include phenol and n-butyl
alcohol, the latter of which is used
to fractionate amylose from native starch via this complex formation (Takeo and
Kuge 1968). Acetic (ethanoic) acid, the active ingredient in vinegar, shares
this amphiphilic quality and may therefore participate in helical complex
formation with starch chains. However, the physiological relevance of this
interaction with respect to the antiglycaemic effects of vinegar is not known.
The aim of this study was to determine whether the
postprandial antiglycaemic effect exerted by vinegar may be attributed to
formation of a double-helical amylose inclusion complex with acetic acid, which
may increase resistant starch formation and decrease the rate of digestion in vivo, or a physiological mechanism
not presently well understood. Two experiments are described: in Part I, the
effects of a physiologically-relevant dose of acetic acid on digestibility of
starch paste in the presence or absence of amylose are described. Part II
describes the digestibility of a prepared starch-acetic acid complex with up to
10 wt% acetic acid (dsb) made by precooking and predrying of starch paste prior
to hydrolysis. Digestibility of freshly-prepared starch pastes are also
described in Part II.
Materials
Normal corn starch (NC) and waxy corn starch (WC) were from
Cargill and American Maize Products, respectively. Acetic acid, sodium acetate,
calcium chloride, sodium phosphate (mono- and di-basic) were from Fisher
Scientific (Waltham , MA St.
Louis , MO Wicklow , Ireland
Part I
Experimental conditions in Part I were chosen to reflect
realistic intakes of acetic acid as vinegar with meals and normal behaviour of
humans in adding vinegar to meals. NC containing 28.6% apparent amylose WC
containing 0.26% apparent amylose were selected in this study to compare the
influence of amylose on starch digestibility in the presence of acetic acid.
Briefly, 0.5 g (dsb) NC and WC were measured into 50-mL centrifuge tubes fitted
with screw caps (Corning, NY) to which 7.5 mL sodium phosphate buffer (100 mM,
pH 6.9, 0.02% NaN3, 0.005% CaCl2) was added with a stir
bar. Samples were cooked in a > 95 °C water bath for 10 min with stirring to
form a paste. Following cooking, 2.5 mL of distilled, deionized (DI) water, 5%
acetic acid solution (v/v), or 10% acetic acid solution (v/v) were added to
starch pastes and mixed thoroughly with a vortex mixer. Concentrations of 5 and
10% acetic acid solutions prior to addition to starch pastes were 0.874 and
1.749 M, respectively; approximate acetic acid concentrations after addition to
starch paste samples were 0.219 and 0.437 M, respectively. Samples were capped
and placed in a 37 °C water bath with lateral shaking (80 rpm) for 1 h to allow
for formation of starch-acetic acid complex. Sample pH was adjusted to 6.9
following equilibration using 0.5 M NaOH, and total volume was adjusted to 15
mL with DI water. Samples were returned to the 37 °C water bath to equilibrate
for 30 min. Englyst method enzyme solution (2.5 mL, see below) was added to
samples, and 250 μL sample aliquots were
removed to tubes containing 10 mL 66% ethanol at 0, 20, 60, and 120 min for
determination of rapidly digestible starch (RDS), slowly digestible starch
(SDS), and resistant starch (RS) fractions.
The Englyst method for RS determination was used in this
study with minor modifications because this method is designed to emulate the
actual human digestive process as closely as can be achieved in vitro (Englyst and others 1992). To
make up enzyme solution added to samples, 3.0 g of porcine pancreatin was
suspended in 20 mL of distilled water in each of 4 50-mL centrifuge tubes using
a vortex mixer and was stirred for 10 min with a stir bar. Tubes containing
pancreatin slurry were centrifuged at 1500 g using a Sorvall RC 5B Plus
laboratory centrifuge (Fisher Scientific) for 10 min and 13.5 mL of the
supernatant from each tube was removed to a clean beaker. Concurrent with this
preparation, 3.15 mL of amyloglucosidase was suspended in 3.6 mL of distilled
water. Diluted amyloglucosidase (6 mL) was added with 4 mL of distilled water
to pancreatin supernatant and mixed. Enzyme solution was prepared immediately
prior to use each day.
Part II
The protocol used in Part II was designed to maximize
helical complex interaction between starch chains and acetic acid molecules
according to the method of Ai and others (2013). NC and WC (4.0 g, db) were
cooked with 3x of water (w/w) and 0, 5, or 10% acetic acid (w/w, dsb) in a ≥ 95
°C water bath for 8 min with constant manual stirring to ensure that starch was
completely gelatinized. Starch pastes were dried overnight in a 50 °C
convection oven and were ground to a powder using a coffee grinder (Mr. Coffee®,
Sunbeam Products, Cleveland ,
OH
Ground starch pastes (0.5 mg, dsb) of NC and WC cooked
without acetic acid as control (NC-C and WC-C, respectively), 5% acetic acid
(NC-5 and WC-5, respectively), and 10% acetic acid (NC-10 and WC-10) were
measured into 50-mL centrifuge tubes with 15 mL sodium phosphate buffer (100
mM, pH 6.9, 0.02% NaN3, 0.005% CaCl2). Tubes containing
ground starch paste and phosphate buffer were homogenized using a T25
ULTRA-TURRAX® benchtop homogenizer (IKA Works, Staufen, Germany) at 11000 rpm for 45 s
and were placed in a 37 °C water bath with shaking (80 rpm) for 30 min to
equilibrate. Samples were hydrolyzed using the Englyst method procedure for RS
content determination described in Part I.
Results and Discussion
Treatment of NC and WC starch pastes with aqueous acetic acid solutions
representing vinegar (5% acetic acid) and twice-concentrated vinegar (10% acetic
acid) after cooking to represent addition of vinegar to starchy ingesta in the
stomach did not significantly affect starch digestibility compared with
controls (Figure 1A). Proportions of RDS, SDS, and RS fractions determined in
starch pastes were generally not significantly different, and no apparent
dose-dependent effect of acetic acid on starch fractions was observed. Several
reasons may exist for this apparent non-interaction of acetic acid with starch
pastes. Equilibration time of 1h may not have been sufficient to induce helical
complex formation with acetic acid, although this equilibration time was chosen
to reflect residence time of chyme in the stomach prior to emptying into the
duodenum for amylolytic digestion to occur. Molar concentrations of acetic acid
in the final starch paste may have been insufficient to maximize helical
complex formation. Therefore, the
influence of two levels of acetic acid addition on digestibility of predried
starch pastes were investigated.
Digestibility of precooked, dried, and ground (PDG)
starch pastes containing 0, 5, and 10% acetic acid (dsb) during cooking were significantly
different (Figure 1B). Digestion rates of the PDG starch pastes were lower than
that of starch pastes with the addition of aqueous acetic acid solution,
indicating that the drying process increased the SDS and RS fractions and
decreased the proportion of RDS fraction (Table 2B) relative to that of undried
starch pastes, most notably in the control treatments without addition of
acetic acid. This change in the proportion of starch fractions is attributed to
retrogradation of starch chains during overnight drying. This interpretation is
supported by the observation that digestibility of NC pastes, which contain
about 30% amylose, were uniformly lower than that of WC pastes, which contain
no amylose. Amylose content is a strong determinant of starch digestibility
rate due to the formation of double-helical crystalline complexes of linear
starch chains, resistant starch type 3 (RS3) during retrogradation.
Digestibility of freshly-prepared starch pastes containing
similar amounts of acetic acid during cooking to investigate effects of
starch-acetic acid complex formation in the absence of retrogradation during
drying and storage (Figure 1C). Digestibility of NC and WC pastes for control,
5%, and 10% acetic acid treatments were not significantly different between
starches, but digestibility also increased with increasing concentrations of
acetic acid in a dose-dependent fashion. This result also appears to support
the interpretation that hydrolysis of starch chains occurred in preference to
RS-generative helical complex formation in acetic acid treatment of starch
pastes cooked with a minimum of water.
 |
| A |
 |
| B |
 |
| C |
Figure 1 (Above). (A) Hydrolysis of starch pastes by porcine
pancreatin up to 120 min after addition of water (control), 5% acetic acid (AA)
(0.219 M), or 10% AA (0.437 M); (B) hydrolysis of precooked, dried starch
pastes with 0, 5, or 10% AA (dsb) by porcine pancreatin up to 120 min. (Author's Aside: Also including various manglings by an older version of Microsoft Office.)
PDG starch pastes were digested more quickly in a
dose-dependent fashion upon addition of acetic acid for both NC and WC pastes
(Figure 1B). Proportions of SDS and RS fractions in PDG pastes generally
decreased and RDS generally increased upon addition of acetic acid, although
SDS was not significantly different in NC PDG starch pastes except for the
addition of 10% acetic acid (Table 2B). Relative proportions of SDS and RS
decreased and RDS increased in freshly-prepared starch pastes in a similar
dose-dependent fashion upon cooking with acetic acid (Table 3B). Addition of
acetic acid would be expected to result in an increase in SDS and/or RS
fractions and a decrease in the overall digestibility rate if helical complex
formation with acetic acid was a predominant process in the lowering of
postprandial glycaemia by vinegar. The observation that addition of vinegar
instead decreases these starch fractions and increases digestibility rate
suggests that acid hydrolysis of starch chains during gelatinization is
favoured over complex formation. Acid hydrolysis of starch chains is expected
to increase digestibility by lowering average DP of starch chains and inhibit
recrystallization of amylose and amylopectin branch chains necessary to
formation of RS during drying. Based on these results, we conclude that
formation of a starch-acetic acid complex is not a significant contributing
factor in the lowering of postprandial glycaemia by pre- or co-ingestion of
vinegar.
Conclusions
Starch digestibility does not appear to be influenced by formation
of a complex with acetic acid when confounding factors (e.g. pH) are minimized.
Digestibility of starch pastes cooked with acetic acid to maximize complex
formation was increased, which suggests that acid hydrolysis predominated over
formation of starch-acetic acid complex. These results appear to implicate an
alternative mechanism for the lowering of postprandial glycaemia due to vinegar
ingestion such as reduced rates of gastric emptying or hitherto unexplored
hormonal influences of acetic acid ingestion.
Works Cited
Ai, Y.; Hasjim, J.; Jane, J. Effects of lipids on enzymatic
hydrolysis and physical properties of starch. Carbohydrate Polymers. 2013,
92, 130 – 137
Ebihara, K.; Nakajima, A. Effect of acetic acid and vinegar
on blood glucose and insulin responses to orally administered sucrose and
starch. Agric. Biol. Chem. 1988, 52, 1311 – 1312
Englyst, H. N.; Kingman, S. M.; Cummings, J. H.
Classification and measurement of nutritionally important starch fractions. Eur. J. Clin Nutr. 1992, 46, S33 – S50
Johnston, C. S.; Kim, C. M.; Buller, A. J. Vinegar Improves
Insulin Sensitivity to a High-Carbohydrate Meal in Subjects with Insulin
Resistance or Type 2 Diabetes. Diabetes
Care. 2004, 27, 1, 281 – 282
Kuge, T.; Takeo, K. Complexes of Starchy Materials with
Organic Compounds. Part I: Affinity Observed by Gas Chromatography. Agric. Biol. Chem. 1968, 32, 6, 753 – 758
Liatis, S.; Grammatikou, S.; Poulia, K.-A.; Perrea, D.;
Makrilakis, K.; Diakoumopolou, E.; Katsilambros, N. Vinegar reduces
postprandial hyperglycaemia in patients with type II diabetes when added to a
high, but not to a low, glycaemic index meal. Eur. J. Clin Nut. 2010,
64, 727 – 732
Östman, E.; Granfeldt, Y.; Persson, L.; Björck, I.
Vinegar supplementation lowers glucose and insulin responses and increases
satiety after a bread meal in healthy subjects. Eur. J. Clin. Nutr. 2005,
59, 983 – 988
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